Crosstalk between p38 MAPK and GR Signaling.

The p38 MAPK is a signaling pathway important for cells to respond to environmental and intracellular stress. Upon activation, the p38 kinase phosphorylates downstream effectors, which control the inflammatory response and coordinate fundamental cellular processes such as proliferation, apoptosis, and differentiation. Dysregulation of this signaling pathway has been linked to inflammatory diseases and cancer. Secretion of glucocorticoids (GCs) is a classical endocrine response to stress. The glucocorticoid receptor (GR) is the primary effector of GCs and plays an important role in the regulation of cell metabolism and immune response by influencing gene expression in response to hormone-dependent activation. Its ligands, the GCs or steroids, in natural or synthetic variation, are used as standard therapy for anti-inflammatory treatment, severe asthma, autoimmune diseases, and several types of cancer. Several years ago, the GR was identified as one of the downstream targets of p38, and, at the same time, it was shown that glucocorticoids could influence p38 signaling. In this review, we discuss the role of the crosstalk between the p38 and GR in the regulation of gene expression in response to steroids and comprehend the importance and potential of this interplay in future clinical applications.

The long noncoding RNA NEAT1 and nuclear paraspeckles are up-regulated by the transcription factor HSF1 in the heat shock response.

The long noncoding RNA (lncRNA) NEAT1 (nuclear enriched abundant transcript 1) is the architectural component of nuclear paraspeckles, and it has recently gained considerable attention as it is abnormally expressed in pathological conditions such as cancer and neurodegenerative diseases. NEAT1 and paraspeckle formation are increased in cells upon exposure to a variety of environmental stressors and believed to play an important role in cell survival. The present study was undertaken to further investigate the role of NEAT1 in cellular stress response pathways. We show that NEAT1 is a novel target gene of heat shock transcription factor 1 (HSF1) and is up-regulated when the heat shock response pathway is activated by sulforaphane (SFN) or elevated temperature. HSF1 binds specifically to a newly identified conserved heat shock element in the NEAT1 promoter. In line with this, SFN induced the formation of NEAT1-containing paraspeckles via an HSF1-dependent mechanism. HSF1 plays a key role in the cellular response to proteotoxic stress by promoting the expression of a series of genes, including those encoding molecular chaperones. We have found that the expression of HSP70, HSP90, and HSP27 is amplified and sustained during heat shock in NEAT1-depleted cells compared with control cells, indicating that NEAT1 feeds back via an unknown mechanism to regulate HSF1 activity. This interrelationship is potentially significant in human diseases such as cancer and neurodegenerative disorders.

Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress.

BACKGROUND: The transcription factor PAX6 is expressed in various cancers. In anaplastic astrocytic glioma, PAX6 expression is inversely related to tumor grade, resulting in low PAX6 expression in Glioblastoma, the highest-grade astrocytic glioma. The aim of the present study was to develop a PAX6 knock out cell line as a tool for molecular studies of the roles PAX6 have in attenuating glioblastoma tumor progression. METHODS: The CRISPR-Cas9 technique was used to knock out PAX6 in U251 N cells. Viral transduction of a doxycycline inducible EGFP-PAX6 expression vector was used to re-introduce (rescue) PAX6 expression in the PAX6 knock out cells. The knock out and rescued cells were rigorously characterized by analyzing morphology, proliferation, colony forming abilities and responses to oxidative stress and chemotherapeutic agents. RESULTS: The knock out cells had increased proliferation and colony forming abilities compared to wild type cells, consistent with clinical observations indicating that PAX6 functions as a tumor-suppressor. Cell cycle distribution and sensitivity to H2O2 induced oxidative stress were further studied, as well as the effect of different chemotherapeutic agents. For the PAX6 knock out cells, the percentage of cells in G2/M phase increased compared to PAX6 control cells, indicating that PAX6 keeps U251 N cells in the G1 phase of the cell cycle. Interestingly, PAX6 knock out cells were more resilient to H2O2 induced oxidative stress than wild type cells. Chemotherapy treatment is known to generate oxidative stress, hence the effect of several chemotherapeutic agents were tested. We discovered interesting differences in the sensitivity to chemotherapeutic drugs (Temozolomide, Withaferin A and Sulforaphane) between the PAX6 expressing and non-expressing cells. CONCLUSIONS: The U251 N PAX6 knock out cell lines generated can be used as a tool to study the molecular functions and mechanisms of PAX6 as a tumor suppressor with regard to tumor progression and treatment of glioblastoma.

Detection of drug-resistant clones in chronic myelogenous leukemia patients during dasatinib and nilotinib treatment.

BACKGROUND: Imatinib effectively inhibits the tyrosine kinase activity conferred by the BCR-ABL gene [fusion gene of BCR (breakpoint cluster region) and ABL1 (c-abl oncogene 1, receptor tyrosine kinase)] and thereby appreciably improves outcomes for chronic myelogenous leukemia (CML). A small percentage of patients relapse because of the proliferation of escape clones; such relapses can be treated with second-generation drugs. Early detection and monitoring of resistant clones may provide clinical benefit. We describe the development and testing of a new approach for quantitative monitoring of CML resistance. METHODS: We designed mutation-specific assays that use hydrolysis probes and an array of allele-specific primers containing nucleotides mismatched at various positions. All assays were tested with plasmids containing corresponding mutant or wild-type sequences, allowing identification of optimal assays for specific and effective amplification of the target template. Clinical samples were then used to compare the results of selected assays with those of standard genotyping. RESULTS: We used a modified amplification refractory mutational system approach and testing with plasmid constructs to design assays that allowed highly selective detection of resistance for all target mutations. By taking advantage of single-step performance and high PCR efficiency, we were able to quantitatively track the absolute amount of resistance conferred by a specific mutation over 4 orders of magnitude. Moreover, we designed an integrated test for dasatinib resistance that uses multiple primers simultaneously. CONCLUSIONS: These single-step, closed-tube assays specifically target mutations associated with resistance to dasatinib or nilotinib. Compared with standard genotyping, such biased genotyping improves the detection of resistance or alternative features via quantitative analysis of the absolute amount of resistance.

Phosphorylation of steroid receptor coactivator-3 (SRC-3) at serine 857 is regulated by the p38MAPK-MK2 axis and affects NF-κB-mediated transcription.

Steroid receptor coactivator-3 (SRC-3) regulates the activity of both nuclear hormone receptors and a number of key transcription factors. It is implicated in the regulation of cell proliferation, inflammation and in the progression of several common cancers including breast, colorectal and lung tumors. Phosphorylation is an important regulatory event controlling the activities of SRC-3. Serine 857 is the most studied phospho-acceptor site, and its modification has been reported to be important for SRC-3-dependent tumor progression. In this study, we show that the stress-responsive p38MAPK-MK2 signaling pathway controls the phosphorylation of SRC-3 at S857 in a wide range of human cancer cells. Activation of the p38MAPK-MK2 pathway results in the nuclear translocation of SRC-3, where it contributes to the transactivation of NF-kB and thus regulation of IL-6 transcription. The identification of the p38MAPK-MK2 signaling axis as a key regulator of SRC-3 phosphorylation and activity opens up new possibilities for the development and testing of novel therapeutic strategies to control both proliferative and metastatic tumor growth.

A co-operative evaluation of different methods of detecting BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia on second-line dasatinib or nilotinib therapy after failure of imatinib.

BACKGROUND: Various techniques have been employed to detect BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia who are resistant to imatinib. This has led to different reported frequencies of mutations and the finding of a heterogeneous pattern of individual mutations. DESIGN AND METHODS: We compared direct sequencing alone and in combination with denaturing high-performance liquid chromatography and two high-sensitivity allele-specific oligonucleotide polymerase chain reaction approaches for analysis of BCR-ABL mutations in 200 blinded cDNA samples prior to and during second-line dasatinib or nilotinib therapy in patients with chronic myeloid leukemia in whom imatinib treatment had failed. RESULTS: One hundred and fourteen mutations were detected by both direct sequencing alone or in combination with high performance liquid chromatography and 13 mutations were additionally detected by the combined technique. Eighty of 83 mutations (96%) within a selected panel of 11 key mutations were confirmed by both allele-specific oligonucleotide polymerase chain reaction techniques and 62 mutations were identified in addition to those detected by combined liquid chromatography and direct sequencing, indicating the presence and a high prevalence of low-level mutations in this cohort of patients. Furthermore, 125 mutations were detected by only one allele-specific oligonucleotide polymerase chain reaction technique. Pre-existing mutations were traceable 4.5 months longer and emerging clones were detectable 3.0 months earlier by allele-specific oligonucleotide polymerase chain reaction than by direct sequencing together with liquid chromatography. CONCLUSIONS: Our results suggest that denaturing high performance liquid chromatography combined with direct sequencing is a reliable screening technique for the detection of BCR-ABL kinase domain mutations. Allele-specific oligonucleotide polymerase chain reaction further increases the number of detected mutations and indicates a high prevalence of mutations at a low level. The clinical impact of such low-level mutations remains uncertain and requires further investigation. Allele-specific oligonucleotide polymerase chain reaction allows detection of defined mutations at a lower level than does denaturing high performance liquid chromatography combined with direct sequencing and may, therefore, provide clinical benefit by permitting early reconsideration of therapeutic strategies.

MicroRNA-193b-3p represses neuroblastoma cell growth via downregulation of Cyclin D1, MCL-1 and MYCN.

Neuroblastoma is the most common diagnosed tumor in infants and the second most common extracranial tumor of childhood. The survival rate of patients with high-risk neuroblastoma is still very low despite intensive multimodal treatments. Therefore, new treatment strategies are needed. In recent years, miRNA-based anticancer therapy has received growing attention. Advances in this novel treatment strategy strongly depends on the identification of candidate miRNAs with broad-spectrum antitumor activity. Here, we identify miR-193b as a miRNA with tumor suppressive properties. We show that miR-193b is expressed at low levels in neuroblastoma cell lines and primary tumor samples. Introduction of miR-193b mimics into nine neuroblastoma cell lines with distinct genetic characteristics significantly reduces cell growth in vitro independent of risk factors such as p53 functionality or MYCN amplification. Functionally, miR-193b induces a G1 cell cycle arrest and cell death in neuroblastoma cell lines by reducing the expression of MYCN, Cyclin D1 and MCL-1, three important oncogenes in neuroblastoma of which inhibition has shown promising results in preclinical testing. Therefore, we suggest that miR-193b may represent a new candidate for miRNA-based anticancer therapy in neuroblastoma.

The third helix of the homeodomain of paired class homeodomain proteins acts as a recognition helix both for DNA and protein interactions.

The transcription factor Pax6 is essential for the development of the eyes and the central nervous system of vertebrates and invertebrates. Pax6 contains two DNA-binding domains; an N-terminal paired domain and a centrally located homeodomain. We have previously shown that the vertebrate paired-less isoform of Pax6 (Pax6DeltaPD), and several other homeodomain proteins, interact with the full-length isoform of Pax6 enhancing Pax6-mediated transactivation from paired domain-DNA binding sites. By mutation analyses and molecular modeling we now demonstrate that, surprisingly, the recognition helix for specific DNA binding of the homeodomains of Pax6 and Chx10 interacts with the C-terminal RED subdomain of the paired domain of Pax6. Basic residues in the recognition helix and the N-terminal arm of the homeodomain form an interaction surface that binds to an acidic patch involving residues in helices 1 and 2 of the RED subdomain. We used fluorescence resonance energy transfer assays to demonstrate such interactions between Pax6 molecules in the nuclei of living cells. Interestingly, two mutations in the homeodomain recognition helix, R57A and R58A, reduced protein-protein interactions, but not DNA binding of Pax6DeltaPD. These findings suggest a critical role for the recognition helix and N-terminal arm of the paired class homeodomain in protein-protein interactions.

Pax6 regulates the expression of Dkk3 in murine and human cell lines, and altered responses to Wnt signaling are shown in FlpIn-3T3 cells stably expressing either the Pax6 or the Pax6(5a) isoform.

Pax6 is a transcription factor important for early embryo development. It is expressed in several cancer cell lines and tumors. In glioblastoma, PAX6 has been shown to function as a tumor suppressor. Dickkopf 3 (Dkk3) is well established as a tumor suppressor in several tumor types, but not much is known about the regulation of its expression. We have previously found that Pax6 and Pax6(5a) increase the expression of the Dkk3 gene in two stably transfected mouse fibroblast cell lines. In this study the molecular mechanism behind this regulation is looked at. Western blot and reverse transcriptase quantitative PCR (RT-qPCR) confirmed higher level of Dkk3 expression in both Pax6 and Pax6(5a) expressing cell lines compared to the control cell line. By the use of bioinformatics and electrophoretic mobility shift assay (EMSA) we have mapped a functional Pax6 binding site in the mouse Dkk3 promoter. The minimal Dkk3 promoter fragment required for transcriptional activation by Pax6 and Pax6(5a) was a 200 bp region just upstream of the transcriptional start site. Mutation of the evolutionary conserved binding site in this region abrogated transcriptional activation and binding of Pax6/Pax6(5a) to the mouse Dkk3 promoter. Since the identified Pax6 binding site in this promoter is conserved, RT-qPCR and Western blot were used to look for regulation of Dkk3/REIC expression in human cell lines. Six of eight cell lines tested showed changes in Dkk3/REIC expression after PAX6 siRNA knockdown. Interestingly, we observed that the Pax6/Pax6(5a) expressing mouse fibroblast cell lines were less responsive to canonical Wnt pathway stimulation than the control cell line when TOP/FOP activity and the levels of active ß-catenin and GSK3-ß Ser9 phosphorylation were measured after LiCl stimulation.

3T3 cell lines stably expressing Pax6 or Pax6(5a)--a new tool used for identification of common and isoform specific target genes.

Pax6 and Pax6(5a) are two isoforms of the evolutionary conserved Pax6 gene often co-expressed in specific stochiometric relationship in the brain and the eye during development. The Pax6(5a) protein differs from Pax6 by having a 14 amino acid insert in the paired domain, causing the two proteins to have different DNA binding specificities. Difference in functions during development is proven by the fact that mutations in the 14 amino acid insertion for Pax6(5a) give a slightly different eye phenotype than the one described for Pax6. Whereas quite many Pax6 target genes have been published during the last years, few Pax6(5a) specific target genes have been reported on. However, target genes identified by Pax6 knockout studies can probably be Pax6(5a) targets as well, since this isoform also will be affected by the knockout. In order to identify new Pax6 target genes, and to try to distinguish between genes regulated by Pax6 and Pax6(5a), we generated FlpIn-3T3 cell lines stably expressing Pax6 or Pax6(5a). RNA was harvested from these cell lines and used in gene expression microarrays where we identified a number of genes differentially regulated by Pax6 and Pax6(5a). A majority of these were associated with the extracellular region. By qPCR we verified that Ncam1, Ngef, Sphk1, Dkk3 and Crtap are Pax6(5a) specific target genes, while Tgfbi, Vegfa, EphB2, Klk8 and Edn1 were confirmed as Pax6 specific target genes. Nbl1, Ngfb and seven genes encoding different glycosyl transferases appeared to be regulated by both. Direct binding to the promoters of Crtap, Ctgf, Edn1, Dkk3, Pdgfb and Ngef was verified by ChIP. Furthermore, a change in morphology of the stably transfected Pax6 and Pax6(5a) cells was observed, and the Pax6 expressing cells were shown to have increased proliferation and migration capacities.

BCR-ABL isoforms associated with intrinsic or acquired resistance to imatinib: more heterogeneous than just ABL kinase domain point mutations?

Imatinib, a small molecule inhibitor of ABL, PDGFR and C-KIT, has revolutionized treatment of chronic myeloid leukaemia (CML). However, resistance to treatment is of increasing importance and often is due to point mutations in the Abl kinase domain (Abl KD). Here, we analysed clinical outcome and mutation status in two independent Nordic populations (n = 77) of imatinib-resistant CML patients. We detected BCR-ABL transcripts containing point mutations of residues in the P-loop, A-loop and other kinase domain residues in 32 patients (42%). In contrast to previous data, mutations in BCR-ABL were as frequently found in patients with primary resistance (56%) as with secondary resistance (53%). No T315I mutations were found in the study cohort. BCR-ABL splice variants were identified in a significant number of our cases (19%): BCR-ABL transcripts of variable length; a variant fusion transcript joining BCR exon 14 sequences to ABL exon 4; partial, in-frame-deletion of exon 4 due to induction of a cryptic splice site by the L248V and finally, alternative splicing of ABL exon 7 sequences. Though the majority of splice variants observed in this study do not encode functional proteins, alternative splicing appears to represent a common phenomenon in the biology of CML. We conclude that Abl KD point mutations represent a major mechanism of imatinib resistance. Other sequence irregularities were also detected, but their significance in conferring resistance is unclear. Diagnostic strategies looking for imatinib-resistant clones should be designed to detect a broader profile of BCR-ABL variants than just point mutations.

Reversion of B cell commitment upon loss of Pax5 expression.

The transcription factor Pax5 is essential for initiating B cell lineage commitment, but its role in maintaining commitment is unknown. Using conditional Pax5 inactivation in committed pro-B cells, we demonstrate that Pax5 is required not only to initiate its B lymphoid transcription program, but also to maintain it in early B cell development. As a consequence of Pax5 inactivation, previously committed pro-B cells regained the capacity to differentiate into macrophages in vitro and to reconstitute T cell development in vivo in RAG2-/- mice. Hence, Pax5 expression is continuously required to maintain B cell lineage commitment, because its loss converts committed pro-B cells into hematopoietic progenitors with multilineage potential.